The above amidated peptides were synthesized via solid-phase peptide synthesis on a CEM Discover Microwave Peptide Synthesizer (Matthews, NC) using the Fmoc-protection strategy on Rink Amide-ChemMatrix resin (0.5 mmol/g). For coupling reactions, amino acids (5 eq) were activated with N,N′-diisopropylcarbodiimide (DIC, 5 eq, Oakwood Chemical)/Oxyma (5 eq, Oakwood Chemical) and heated to 90 °C for 2 min while bubbling with nitrogen gas in N,N-dimethylformamide (DMF, Oakwood Chemical). Fmoc deprotection was carried out with 20% piperidine (Sigma-Aldrich) in DMF supplemented with 0.1 M 1-hydroxybenzotriazole hydrate (HOBt, Oakwood Chemical) at 90 °C for 1 min while bubbling with nitrogen gas. The H3 Cleavage from the resin was performed with 92.5% TFA, 2.5% triisopropylsilane (TIS, Sigma-Aldrich), 2.5% 1,2-ethanedithiol (EDT, Sigma-Aldrich), and 2.5% H2O for 2 h at 25 °C. The crude peptide was then precipitated by the addition of a 10-fold volume of cold ether and centrifuged at 4000 RCF for 10 min at 4 °C. The pellet was resuspended in Solvent A and purified via preparative RP-HPLC using a linear gradient from 0 to 30% Solvent B over 30 min. Fractions were analyzed on analytical RP-HPLC and ESI-MS and those containing pure product ( > 95%) were pooled, lyophilized, and stored at –80 °C.
1 2 ethanedithiol edt
Manufactured by Merck Group
Sourced in United States
1,2-ethanedithiol (EDT) is a chemical compound with the molecular formula C₂H₆S₂. It is a colorless, odorless liquid with a boiling point of 168°C. EDT's core function is as a chemical reagent used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Triisopropylsilane tis, by Merck Group (3 mentions)
Acetonitrile, by Merck Group (2 mentions)
Trifluoroacetic acid tfa, by Merck Group (2 mentions)
Piperidine, by Merck Group (1 mentions)
Fmoc f oh, by Merck Group (1 mentions)
Fmoc r pbf oh, by Merck Group (1 mentions)
Anhydrous methanol, by Merck Group (1 mentions)
Anhydrous acetonitrile, by Merck Group (1 mentions)
Anhydrous dimethyl sulfoxide dmso, by Merck Group (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine dppc, by Avanti Polar Lipids (1 mentions)
1 4 dithiothreitol dtt, by Merck Group (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n maleimide polyethylene glycol 2000, by Avanti Polar Lipids (1 mentions)
Dspe peg mal, by Avanti Polar Lipids (1 mentions)
Dl dithiothreitol dtt, by Merck Group (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Diethyl ether, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid tfa, by Thermo Fisher Scientific (1 mentions)
N n diisopropylethylamine diea, by Thermo Fisher Scientific (1 mentions)
2 1h benzotriazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophosphate hbtu, by Peptide Institute (1 mentions)
Hexane, by Merck Group (1 mentions)
12 protocols using 1 2 ethanedithiol edt
1
Solid-Phase Synthesis of Amidated Peptides
2
Solid-Phase Synthesis of Peptide Sequences
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[RF]4 and
P[RF]4 sequences were synthesized using a solid-phase Fmoc
strategy.24 (link) All chemicals were of analytical
or HPLC grades. The protected amino acids, (Fmoc-F-OH), (Fmoc-R(Pbf)-OH),
and (Fmoc-P-OH), 1,3-diisopropylcarbodiimide/N-hydroxybenzotriazole
(DIC/HOBt), trifluoroacetic acid (TFA), anisole, thioanisole, dichloromethane
(DCM), dimethylformamide (DMF), 1-methyl-2-pyrrolidinone (NMP), and
1,2-ethanedithiol (EDT) were purchased from Sigma-Aldrich (St. Louis,
MO). Wang resin with 100–200 mesh size was purchased from Advanced
Chemtech (Louisville, KY), with a substitution degree of 0.55 mmol·g–1 and with the first amino acid coupled to the polymeric
support. The protecting group was removed by reaction with 20% of
4-methylpiperidine in dimethylformamide for 30 min. Coupling was carried
out in 5.0 fold excess of DIC/HOBt in DCM/DMF (1:1, v:v). The reactions were monitored using the Kaiser
ninhydrin test.25 (link) The dry-protected resin
was exposed to 90% trifluoroacetic acid, 5.0% thioanisole, 3.0% 1,2-ethanedithiol,
and 2.0% anisole to remove all the protecting groups. After this,
the material was lyophilized and analyzed on a liquid-chromatography
electrospray ionization mass spectrometer, LC-ESI-MS, yielding [RF]4 (MM + H) = 1232.6 (calculated = 1231.6) and P[RF]4 (MM + H) = 1328.7 (calculated = 1327.7).22 (link) The molecular structures are showed inFigure 1 .
P[RF]4 sequences were synthesized using a solid-phase Fmoc
strategy.24 (link) All chemicals were of analytical
or HPLC grades. The protected amino acids, (Fmoc-F-OH), (Fmoc-R(Pbf)-OH),
and (Fmoc-P-OH), 1,3-diisopropylcarbodiimide/N-hydroxybenzotriazole
(DIC/HOBt), trifluoroacetic acid (TFA), anisole, thioanisole, dichloromethane
(DCM), dimethylformamide (DMF), 1-methyl-2-pyrrolidinone (NMP), and
1,2-ethanedithiol (EDT) were purchased from Sigma-Aldrich (St. Louis,
MO). Wang resin with 100–200 mesh size was purchased from Advanced
Chemtech (Louisville, KY), with a substitution degree of 0.55 mmol·g–1 and with the first amino acid coupled to the polymeric
support. The protecting group was removed by reaction with 20% of
4-methylpiperidine in dimethylformamide for 30 min. Coupling was carried
out in 5.0 fold excess of DIC/HOBt in DCM/DMF (1:1, v:v). The reactions were monitored using the Kaiser
ninhydrin test.25 (link) The dry-protected resin
was exposed to 90% trifluoroacetic acid, 5.0% thioanisole, 3.0% 1,2-ethanedithiol,
and 2.0% anisole to remove all the protecting groups. After this,
the material was lyophilized and analyzed on a liquid-chromatography
electrospray ionization mass spectrometer, LC-ESI-MS, yielding [RF]4 (MM + H) = 1232.6 (calculated = 1231.6) and P[RF]4 (MM + H) = 1328.7 (calculated = 1327.7).22 (link) The molecular structures are showed in
3
Synthesis and Characterization of Lead Sulphide Quantum Dots
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After synthesis protocol customisation, high-quality lead sulphide (PbS) QDs with various diameters were tested and procured from Quantum Solutions (www.quantum-solutions.com ). Dimethyl sulfoxide (DMSO, anhydrous, 99%, Sigma-Aldrich), methanol (anhydrous, 99%, Sigma-Aldrich), acetonitrile (anhydrous, 99%, Sigma-Aldrich), 1,2-ethanedithiol (EDT, Sigma-Aldrich), ethylenediamine (EDA, Nacalai Tesque), 1-Ethyl-3-methylimidazolium bis(trifluoro-methylsulfonyl)imide (EMIM-TFSI, Kanto Chemical Co., Inc.) were procured as solvents, subphases, and ionic liquid gates, which were always stored inside N2 gloveboxes.
4
Labeling Cells with ReAsH or FlAsH
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Cells were transfected with the appropriate C4-tag containing plasmids. Cells were cultured on glass coverslips, rinsed with pre-warmed 1× PBS buffer and labeled for 30 min with pre-warmed DMEM containing 1 μM ReAsH or FlAsH (kindly provided by H. Overkleeft, University Leiden, The Netherlands) and 10 μM 1,2-ethanedithiol (EDT, Sigma-Aldrich) at 37 °C. After staining cells were washed twice with 1× PBS for subsequent analysis.
5
PEGylated Liposomal Doxorubicin Synthesis
6
Fluorescent Protein Labeling and Internalization
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Methods were adapted from Taguchi et al. [47 (link)]. Labeling media consisted of 1.4 uM FlAsH reagent (sold under the trade name “Lumio” by Invitrogen), 1 mM 1,2-ethanedithiol (EDT) (Sigma) and 20 mM DL-Dithiothreitol (DTT) (Sigma) incubated together in the dark at room temperature in HBSS for 10 min. SN56 cells were washed and labeled on ice for 3 min. After labeling, cells were washed 2x with room temperature 250 uM EDT in HBSS. The final wash was done with warm HBSS and the cells were allowed to internalize at 37 °C for 15 min. Following internalization, cells were fixed for 15 min in 4 % PFA and imaged on an LSM510 confocal microscope (Carl Zeiss). For FlAsH and anti-HA combination labeling, following treatment with FlAsH labeling media, cells were treated with 1:100 Alexa Fluor-647 Zenon-tagged anti-HA antibodies for 30 min following FlAsH labeling. After labeling with Zenon, cells were washed 2x in warm HBSS and allowed to internalize for 15 min before being fixed.
7
Peptide Synthesis Reagents Protocol
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2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was obtained from Peptides International (Louisville, Kentucky). 1-[Bis (dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxid hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) were obtained from Applied Biosystems (Foster City, California). Piperidine was from Spectrum (Gardena, California). N,N-Dimethylformamide (DMF), methylene chloride (DCM), methanol, diethyl ether, and trifluoroacetic acid (TFA) were from Thermo Fisher Scientific (Pittsburg, Pennsylvania). Triisopropylsilane (TIS) and 1,2-Ethanedithiol (EDT) were from Sigma-Aldrich (Saint Louis, Missouri). HPLC-grade acetonitrile (ACN), amino acids, and TentaGel resin (NovaSyn TGR) were from EMD Millipore (Billerica, Massachusetts). Side chain protecting groups were: Arg (Pbf), Asn (Trt), Asp (OtBu), Cys (Trt), Gln (Trt), Glu (OtBu), His (Trt), Lys (Boc), Ser (tBu), Thr (tBu), Trp (Boc), and Tyr (tBu).
8
Synthesis of Colloidal Nanocrystals
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Indium acetate (In(OAc), 99.99%, Sigma‐Aldrich), oleic acid (OA, 90%), hexane (95% anhydrous, Sigma‐Aldrich), 2‐methyltetrahydrofuran (2‐meTHF, 99.0% anhydrous, Sigma‐Aldrich), 2‐methylanisole (2‐MA, 99.0%, Sigma‐Aldrich), toluene (99.5%, Sigma‐Aldrich), 1,2‐ethanedithiol (EDT, 99.0%, Sigma‐Aldrich), ethanethiol (ET, 97%, Sigma‐Aldrich), chlorobenzene (CB, 99.8% anhydrous, Sigma‐Aldrich), acetonitrile (ACN, 99.5%, Sigma‐Aldrich), ethyl acetate (EA, 99.5%, Sigma‐Aldrich), chloroform (CF, 99.8%, Sigma‐Aldrich), butylamine (BTA, 99.5%, Sigma‐Aldrich), benzoic acid (BA, 99.5%, Sigma‐Aldrich), nitrosyl tetrafluoroborate (NOBF4, 95%, Sigma‐Aldrich), and dioctylamine (DOA, 97%, Sigma‐Aldrich) were purchased and used as received. The compounds 1‐octadecene (ODE, 90%, Alfa Aesar), octane (98%, Alfa Aesar), and 3‐mercaptopropionic acid (MPA, 99%, Alfa Aesar) were purchased and used without further purification. 1‐butanol (BuOH, 99.8%) was purchased from Junsei chemical, ME (99%) was purchased from Daejung, and Tris(trimethylsilyl)arsine ((TMSi)3As, 99%) was purchased from JSI Silicone and was distilled before use.
9
Peptide Synthesis Protocol
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All reagents were purchased from commercial sources and used as received. N-α-Fmoc amino acids were purchased from CreoSalus or Novabiochem. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), N,N′-diisopropylcarbodiimide (DIC) were purchased from Chem-Impex. N,N-Dimethylformamide (DMF) was purchased from EMD Millipore. To each DMF bottle was added an AldraAmine trapping packet (Sigma-Aldrich) to minimize the accumulation of water and amine impurities. N,N-diisopropylethylamine (DIPEA), 4-(dimethylamino)pyridine (DMAP), piperidine, trifluoroacetic acid, triisopropylsilane, acetonitrile and 1,2-ethanedithiol (EDT) were purchased from Sigma-Aldrich. HMPB-ChemMatrix polyethylene glycol resin with a loading of ca. 0.5 mmol/g was purchased from Pcas Biomatrix.
10
Peptide Synthesis and Purification Protocol
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A Tribute peptide synthesiser (Protein Technologies, Inc, USA) was utilised to synthesise the mature peptide. After synthesis, the peptide was transferred to a 50-ml round-bottomed flask. Twenty-five ml per gram of mixture cleavage solution (94 % Trifluoroacetic acid (TFA) (Sigma-Aldrich, USA), 2 % dd H2O, 2 % thioanisole (TIS) (Sigma-Aldrich, USA) and 2 % 1–2 ethanedithiol (EDT) (Sigma-Aldrich, USA)) were added into the round-bottomed flask. The round-bottomed flask was stirred with a rotor in the fume hood for 2 h. After cleavage, the mixture solution was filtered by a Buchner funnel, and 3 ml of dichloromethane (DCM) were used to wash (Sigma-Aldrich, USA) three times. Diethyl ether (Et2O) (Aldrich, USA) was added to the mixture to 50 ml in a 50-ml tube. Moreover, the tube was placed at a temperature of −20 °C to precipitate the peptide. After drying in the fume hood, 10 ml of HPLC buffer A (0.5 ml TFA/999.5 ml H2O) were used to dissolve the peptide, and then buffer B (0.5 ml TFA/199.5 ml H2O/800 ml acetonitrile (Sigma-Aldrich, USA)) 5 ml. The tube was put in an Alpha 1–2 LD plus freeze dryer (CHRIST, Germany) for 48–50 h. The dry peptide was then stored at −20 °C, prior to further analyses.
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